G-quadruplexes are non-canonical nucleic acid structures, formed by the stacking of G-tetrads and stabilized by cations (Phan, A T (2010) FEBS Journal, 277, 1107). Guanine-rich sequences, found ubiquitously in the genome of various species including the human genome, are able to fold into G-quadruplexes. Interestingly, these sequences are non-randomly distributed; they are localized in specific parts of the genome including the telomeres and promoter regions (Todd, A. K et al. (2005) Nucleic Acids Res, 33, 2901; Huppert, J. L. and Balasubramanian, S. (2005) Nucleic Acids Res, 33, 2908; Huppert, J. L. (2008) Biochimie, 90, 1140).
For a long time, these structures were believed to have no relevant contribution to biological processes. However, during the past decade strong evidences were found and pointed to significant roles of G-quadruplex structures in biological processes. In fact, it has been now established that G-quadruplexes are involved in essential cellular functions such as transcription, replication and recombination (Piazza, A et al. (2010) NucleicAcidsRes, 38, 4337; Biffi, G et al. (2013) Nat Chem, 5, 182; Rodriguez, R et al. (2012) Nat ChemBiol, 8, 301). For example, the formation and stabilization of G-quadruplexes has been shown to promote genomic instability of mini-satellites in yeast cells (Piazza, A et al. (2010) Nucleic Acids Res, 38, 4337).
It has been shown that formation and stabilization of G-quadruplexes in telomeres regions, in the promoters of oncogenes, or in the 5′-UTR regions of pre-mRNA oncogenes has anti-cancer effects (Sun, D. et al. (1997) J Med Chem 40, 2113; Mergny J. L., Helene C. (1998) Nat Med. 4, 1366; Balasubramanian, S and Neidle, S (2009) CurrOpinChemBiol, 13, 345). These recent findings stimulated the development of synthetic compounds meant to induce/stabilize G-quadruplex structures. For example, Telomestatin (Shin-ya, K et al. (2001) J Am ChemSoc, 123, 1262) and Phen-DC3 (De Cian et al. (2007) J Am ChemSoc, 129, 1856) are among the most relevant compounds, which bind to G-quadruplexes with a high affinity (with a dissociation constant Kd in the nanomolar range) and a good selectivity against duplex and single-stranded nucleic acids.
G-quadruplex structures are highly diverse in regard of relative strand orientations and loop types, resulting in different topologies including a (i) parallel-type in which four strands point in the same direction; an (ii) hybrid “3+1” type in which three strands point in one direction and the fourth strand points in the opposite one; (iii) an antiparallel-type in which two strands point in one direction and two strands point in the opposite direction. Those topologies lead to different structural molecular shapes, with various loops and grooves of different size and accessibility (Phan, A T (2010) FEBS Journal, 277, 1107). The structural polymorphism of G-quadruplexes depends on their nucleotide sequences and the environmental conditions. In the human genome, G-rich sequences are scattered in different regions of the chromosomes and can form different possible G-quadruplex topologies. So far, most of G-quadruplex binders do not present selectivity against different G-quadruplex topologies and thus exhibit a wide-range genome effects (Biffi, G. et al. (2013) Nat Chem, 5, 182; Rodriguez, R et al. (2012) Nat ChemBiol, 8, 301).
Recently several proteins have been reported to interact with G-quadruplexes (Fry, M (2007) Front Biosci, 133, 9824; Murat, P et al. (2013) CurrOpin Genet Dev. 31, 22). One of such proteins is Rhau (also named DHX36 or G4R1). Rhau is a human helicase of the DEAH-box family, present in all type of cells (Tran, H et al. (2004) Mol Cell, 13, 101) and associated with different functions, including the formation of stress granules, interchromatin granule clusters (Chalupnikova, K et al. (2008) J BiolChem, 283, 35186) and the degradation of urokinase plasmonigen activator mRNA. Interestingly, studies by Nagamine and colleagues (Creacy, S D et al. (2008) J BiolChem, 283, 34626; Booy, E P et al. (2012) Nucleic Acids Res, 40, 4110) demonstrated that Rhau protein specifically unwind and bind G-quadruplexes nucleic acids. The G-quadruplex binding domain was identified to be in the N-terminal region of the protein, ranging from residue 53 to 105 (termed Rhau55).
Due to the structural diversity of G-quadruplexes, it is an object of the present invention to identify binders that selectively discriminate between different G-quadruplex topologies.